U.S. patent number 7,294,510 [Application Number 10/472,490] was granted by the patent office on 2007-11-13 for process for producing nerve stem cells, motor neurons, and gabaergic neurons from embryonic stem cells.
This patent grant is currently assigned to Japan Science and Technology Corporation. Invention is credited to Hideyuki Okano, Takuya Shimazaki.
United States Patent |
7,294,510 |
Okano , et al. |
November 13, 2007 |
Process for producing nerve stem cells, motor neurons, and
GABAergic neurons from embryonic stem cells
Abstract
The present invention provides a method for producing motor
neurons and GABAergic neurons characterized by including
suspension-culturing embryonic stem cells in the presence or
absence of a protein noggin to form embryoid bodies, selectively
amplifying into neural stem cells from them by suspension culture
in the presence of a fibroblast growth factor and a sonic hedgehog
protein, and then differentiating the same. According to this
method, at least motor neurons and GABAergic neurons can be
systemically and efficiently produced from ES cells. Selective
acquisition of neurons would be applicable to transplant therapy
for amyotrophic lateral sclerosis, Huntington's chorea, Alzheimer's
disease, etc.
Inventors: |
Okano; Hideyuki (Tokyo,
JP), Shimazaki; Takuya (Tokyo, JP) |
Assignee: |
Japan Science and Technology
Corporation (Kawaguchi-shi, JP)
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Family
ID: |
18952665 |
Appl.
No.: |
10/472,490 |
Filed: |
October 3, 2001 |
PCT
Filed: |
October 03, 2001 |
PCT No.: |
PCT/JP01/08703 |
371(c)(1),(2),(4) Date: |
September 30, 2003 |
PCT
Pub. No.: |
WO02/081663 |
PCT
Pub. Date: |
October 17, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040092012 A1 |
May 13, 2004 |
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Foreign Application Priority Data
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Mar 30, 2001 [JP] |
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2001-099074 |
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Current U.S.
Class: |
435/377; 435/365;
435/354; 435/368; 530/399; 435/325 |
Current CPC
Class: |
C12N
5/0623 (20130101); A61P 25/28 (20180101); C12N
5/0619 (20130101); C12N 2506/02 (20130101); C12N
2533/32 (20130101); C12N 2501/41 (20130101); C12N
2501/16 (20130101); C12N 2501/91 (20130101); C12N
2500/46 (20130101); C12N 2500/25 (20130101); C12N
2501/392 (20130101); C12N 2501/155 (20130101); C12N
2501/115 (20130101); A61K 35/12 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); A61K 38/18 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Finley et al. Journal of Neurobiol., Sep. 5, 1999, 40(3):271-87.
cited by examiner .
K.S. O'Shea et al.: "Noggin induces a neural phenotype in ES cells,
which is antagonized by EMP-4" Society for Neuroscience, vol. 24,
Nos. 1-2, p. 1526 1998. cited by other .
Sang-Hun Lee et al.: "Efficient generation of midbrain and
hindbrain neurons from mouse embryonic stem cells" Nature
Biotechnology, vol. 18, No. 6, pp. 675-677 Jun. 2000. cited by
other .
Tsutomu Nohno et al.: "Involvement of the sonic hedgehog genein
chick feather formulation" Biotechnical and Biophysical Research
Communications, vol. 206, No. 1, pp. 33-39 Jan. 5, 1995. cited by
other .
Streit, A., et al., "Chordin regulates primitive streak development
and the stability of induced neural cells, but is not sufficient
for neural induction in the chick embryo," Development 125,
507-519, 1998. cited by other.
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Primary Examiner: Romeo; David S
Assistant Examiner: Gamett; Daniel C
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, P.C.
Claims
The invention claimed is:
1. A method for forming embryoid bodies, which comprises subjecting
embryonic stem cells to suspension culture in the presence of a
noggin protein, wherein said noggin protein is added to the culture
prior to formation of embryoid bodies.
2. A method for producing neural stem cells, which comprises
subjecting embryonic stem cells to suspension culture in the
presence of a noggin protein, to thereby form embryoid bodies, and
subsequently subjecting the embryoid bodies to suspension culture
in the presence of a fibroblast growth factor and a sonic hedgehog
protein to thereby form a culture of neural stem cells.
3. The method according to claim 2, wherein concentration of the
fibroblast growth factor in culture medium is 5 to 50 ng/mL, and
that of the sonic hedgehog protein in culture medium is 1 to 20
nM.
4. A method for producing motor neurons and GABAergic neurons,
which comprises subjecting ES cells to suspension culture in the
presence of a noggin protein, to thereby form embryoid bodies, and
subsequently subjecting the embryoid bodies to suspension culture
in the presence of a fibroblast growth factor and a sonic hedgehog
protein, to thereby induce formation of neural stem cells, and
differentiating the resultant neural stem cells in a
differentiation medium.
5. The method according to claim 4, wherein concentration of the
fibroblast growth factor in culture medium is 5 to 50 ng/mL, and
that of the sonic hedgehog protein in culture medium is 1 to 20
nM.
6. The method according to claim 4, wherein the resultant neurons
are substantially formed of motor neurons and GABAergic
neurons.
7. The method according to claim 5, wherein the resultant neurons
are substantially formed of motor neurons and GABAergic
neurons.
8. The method according to claim 2, wherein the culture of neural
stem cells comprises neurospheres.
Description
TECHNICAL FIELD
The present invention relates to a method for selectively producing
neural stem cells from embryonic stem cells (ES cells) and also to
a method for selectively and efficiently producing motor neurons
and GABAergic neurons.
BACKGROUND OF THE INVENTION
In the central nervous system of a mammal, neural stem cells exist
through the entire life of the individual and contribute to the
growth and homeostasis of the central nervous system by producing a
variety of neurons and glia. Techniques that have recently been
developed aiming at the isolation and culturing of neural stem
cells from the brain of mammals, including humans, are expected to
provide potential applications to the cell transplant therapies for
various types of neurodegenerative diseases and injures. No
appreciable achievement is still reported, however, despite some
attempts exerted to obtain, from the neural stem cells cultured and
amplified in vitro, different types of neurons that can be
generated and differentiated from stem cells under control of
diversified endogenous and exogenous factors, espeGially motor
neurons that can be specifically generated at the initial stage of
embryogenesis.
Accordingly, an object of the present invention is to provide means
for efficiently inducing differentiation of ES cells, which have
the capacity to differentiate into any type of mature cells in an
individual, into neural stem cells maintaining properties of those
cells in the early stage of development. Another object of the
present invention is to provide a technique for selectively
producing a specific type of neuron, such as motor neurons, from
the neural stem cells.
DISCLOSURE OF THE INVENTION
The present inventors have investigated a variety of conditions
under which it is necessary for embryoid bodies to be generated
from ES cells, and for ES cells to differentiate and be induced
into neural stem cells and eventually become the neurons. As a
result, it has been found that the presence of noggin protein has a
particularly important role in the induction of neural stem cells
within embryoid bodies derived from ES cells; the use of a medium
containing a fibloblast growth factor (FGF) and a sonic hedgehog
protein is extremely efficient for amplifying neutral stem cells
emerging in embryoid bodies; and when such neutral stem cells are
differentiated, motor neurons and GABAergic neurons can be produced
selectively and efficiently. Thus, the present invention has been
accomplished on the basis of these findings.
Accordingly, the present invention provides a method for forming
embryoid bodies, characterized by subjecting ES cells to suspension
culture in the presence of noggin protein.
The present invention also provides a method for producing neural
stem cells, characterized by subjecting ES cells to suspension
culture in the presence or absence of noggin protein, to thereby
form embryoid bodies, and subsequently subjecting the embryoid
bodies to suspension culture in the presence of fibroblast growth
factor and sonic hedgehog protein.
The present invention also provides a method for producing motor
neurons and GABAergic neurons, characterized by subjecting ES cells
to suspension culture in the presence or absence of noggin protein,
to thereby form embryoid bodies, and subsequently subjecting the
embryoid bodies to suspension culture in the presence of fibroblast
growth factor and sonic hedgehog protein, to thereby induce neural
stem cells, and differentiate the resultant neural stem cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the relation between days of culturing embryoid bodies
and formation of neurospheres.
FIG. 2 shows an image of immunostaining of neurospheres after
differentiation. The stained region shows expression of
.beta.-III-tubulin, which indicates a neuron.
FIG. 3 shows another image of immunostaining of differentiated
neurospheres by anti-1st-1 and anti-ChAT (choline
acetyltransferase) antibodies which are markers for motor
neurons.
FIG. 4 shows yet another image of immunostaining of differentiated
neurospheres by anti-GAD (glutamic acid decarboxylase) 67
antibody.
FIG. 5 shows an image of immunostaining of differentiated
neurospheres that have undergone subculture. Cells were
immunolabeled by anti-.beta.-III, GFAP, and 04 antibodies.
FIG. 6 shows the percentage of neurons and glia cells after
subculture of neurospheres.
FIG. 7 shows the effect of addition of a noggin protein.
FIG. 8 shows the effect of addition of a sonic hedgehog
protein.
BEST MODE FOR CARRYING OUT THE INVENTION
The ES cells used in the present invention may be those which-have
already been established as cultured cells. For example, ES cell
lines from mice, hamsters, pigs, and humans may be employed.
Specific examples include 129/O1a-mouse-derived ES cells, such as
EB3 and E14tg2. Preferably, the ES cells are subcultured in a GMEM
medium or a similar medium supplemented with serum.
In the formation of embryoid bodies from ES cells, suspension
culture of ES cells in a medium to which noggin protein has been
added is effective for promoting differentiation-inducing
efficiency from ES cells to neural stem cells. The noggin protein
may be a Xenopus noggin protein. Alternatively, full-length cDNA of
Xenopus noggin is transferred to COS7 cells, followed by culturing
to cause transient expression of the noggin protein, and the
resultant supernatant may be used as is. Preferably, the
concentration of the noggin protein in medium is 1 to 50% (v/v) or
thereabouts in terms of the volume of culture supernatant.
Suspension culture of ES cells is performed by use of
serum-containing .alpha.-MEM medium for 4 to 8 days at a
concentration of approximately 1.times.10.sup.5 ES cells/mL.
Examples of useful sera include bovine serum and pig serum. The
serum concentration is 5 to 15%, preferably 8 to 12%. Preferably,
2-mercaptoethanol is added to the .alpha.-MEM medium in such an
amount that achieves a concentration of 0.01 to 0.5 mM,
particularly 0.05 to 0.2 mM. The culturing is preferably performed
in 5% CO.sub.2, at 35-40.degree. C.
It is highly preferred that the noggin protein be added during
formation of embryoid bodies; i.e., during the period from day 0 to
day 6 of culturing.
In order to amplify neural stem cells which have been obtained from
ES cells via the above-prepared embryoid bodies, suspension culture
is performed by use of a neural stem cell amplification medium
containing not only a fibroblast growth factor but also a sonic
hedgehog protein. The addition of sonic hedgehog protein promotes
efficiency of inducing differentiation of neural stem cells to
motor neuron precursors, and also improves multiplication
efficiency of the neural stem cells. Moreover, through subsequent
differentiation culturing, the neural stem cells are in fact
differentiated into motor neurons and GABAergic neurons.
A preferred fibroblast growth factor (FGF) is FGF-2. The FGF
content of the medium is preferably 5 to 50 ng/mL, more preferably
10 to 40 ng/mL. Examples of preferred sonic hedgehog proteins
include mouse sonic hedgehog protein. The sonic hedgehog protein
content of the medium is 1 to 20 nM, preferably 1 to 10 nM.
The medium is preferably a DMEM medium containing, in addition to
the aforementioned components, glucose, glutamine, insulin,
transferrin, progesterone, putrecine, selenium chloride, heparin,
etc. Use of a DMEM:F12 medium is particularly preferred. The
culturing is preferably performed in 5% CO.sub.2, at 35-40.degree.
C., for a period of 7 to 9 days.
Through the above-described suspension culture,
single-cell-derived, aggregated masses of cells, called
neurospheres, are formed.
The thus-obtained neurospheres have originated solely from neural
stem cells, and thus the above-mentioned culture method is proven
to attain very high differentiation efficiency.
When the thus-obtained neural stem cells are cultured in an
ordinary differentiation medium, differentiation into only motor
neurons and GABAergic neurons alone is induced. Here, a preferred
differentiation-inducting medium is a DMEM:F12 medium containing
glucose, glutamine, insulin, transferrin, progesterone, putrecine,
and selenium chloride (in other words, a medium designed for
amplifying neural stem cells but excluding FGF and heparin). In
this medium, sonic hedgehog protein may or may not be present. The
culturing is preferably performed in 5% CO.sub.2, at 35-40.degree.
C., for 5 to 7 days.
Neural cells obtained from ES cells through conventional techniques
of differentiation contain not only neurons but also significant
amounts of glia cells, among other cells. Thus, heretofore, they
have only limited usage value. In contrast, wherein the neurons
obtained by working the present invention are substantially formed
only of motor neurons and GABAergic neurons.
EXAMPLES
The present invention will next be described by way of examples,
which should not be construed as limiting the invention
thereto.
A. Materials and methods
(1) Culture-Passage of Mouse ES Cells and Formation of Embryoid
Bodies
E14tg2a ES cells derived from 129/O1a mice and EB3 ES cells (which
allow selection of undifferentiated ES cells through insertion of
blasticidin-resistant gene to the Oct3/4 locus of E14tg2a) were
subcultured by a routine method in a GMEM medium (Glasgow minimum
essential medium) containing 10% fetal calf serum, nonessential
amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and
1,000 U/mL leukemia inhibitory factor (LIF). The culture conditions
were 5% CO.sub.2 at 37.degree. C. (hereafter, when "culture" is
referred to, these conditions apply).
Formation of embryoid bodies (EBs) from the ES cells was carried
out as follows. Firstly, ES cells were washed with PBS.
Subsequently, the washed cells were treated with 0.25% trypsin--1
mM EDTA, and then the treatment reaction was stopped. The cells
were dissociated by pipetting, and seeded in a bacterial culture
dish filled with .alpha.-MEM medium containing 10% fetal calf serum
and 0.1 mM 2-mercaptoethanol. In the presence or absence of noggin
protein, suspension culture was performed for 4 to 8 days, whereby
EBs were formed. The noggin protein employed was a culture
supernatant of COS7 cells to which full-length cDNA of Xenopus
noggin had been introduced for transitory expression.
(2) Isolation of Neural Stem Cells by Selective Culture of EBs
The EBs formed as described above, together with the culture
liquid, were transferred to a centrifuge tube. The tube was allowed
to stand for 10 minutes, so that the EBs were sedimented at the
bottom. The supernatant was removed, and the EBs were re-suspended
in PBS. The test tube was allowed to stand for 10 minutes again.
The supernatant was removed, and the EBs were re-suspended in a
solution containing 0.25% trypsin and 1 mM EDTA PBS, followed by
incubation at 37.degree. C. for five minutes. The protein
degradation reaction was stopped by use of .alpha.-MEM medium
containing 10% fetal calf serum. The cells were dissociated by
pipetting. The dissociated cells were centrifugally washed with
.alpha.-MEM medium twice, and seeded at a concentration of
5.times.10.sup.4 cells/mL in either of the following mediums
designed for neural stem cell amplification: a 1:1 medium of DMEM
(Dulbecco's modified Eagle's medium) and F12, where the DMEM had
been supplemented with glucose (0.6%), glutamine (2 mM), insulin
(25 .mu.g/mL), transferrin (100 .mu.g/mL), progesterone (20 nM),
putrecine (60 .mu.M), selenium chloride (30 nM), FGF-2 (20 ng/mL),
and heparin (2 .mu.g/mL); or the same medium but further containing
a mouse sonic hedgehog (5 nM), followed by suspension culture for 7
to 9 days, whereby neurospheres (cell clusters derived from a
single cell) were formed. The neurospheres were centrifugally
washed with a differentiation medium containing neither FGF-2 nor
heparin, and the washed cells--in the "as washed" state or after
dissociated through pipetting--were seeded in a culture petri dish
coated with poly-L-ornithine and filled with a differentiation
medium, whereby differentiation is allowed to proceed in the
presence or absence of a sonic hedgehog protein (5 nM) for 5 to 7
days. Separately, the above-obtained neurospheres were again
dissociated into single cells, subcultured in a medium designed for
amplification of neural stem cells, to thereby form secondary
neurospheres. The thus-obtained secondary neurospheres are also
caused to differentiate as described above.
(3) Identification of Differentiated Neurons and Glia Cells Through
Immunostaining
The thus-differentiated neurons and glia cells were identified by a
routine immunostaining method using a fluorescent antibody. Motor
neurons were identified by mouse anti-Isl-1 monoclonal antibody,
goat anti-ChAT polyclonal antibody, and mouse anti-.beta.-III
tublin monoclonal antibody; and GABAergic neurons were identified
by rabbit anti-GAD67 polyclonal antibody. Regarding glia cells,
astrocytes were identified by rabbit anti-GFAP polyclonal antibody,
and oligodendrocytes were identified by mouse anti-04 monoclonal
antibody.
B. Test results
(1) Isolation and Purification of Neural Stem Cells by Selective
Culture of EBs
Firstly, the inventors focused on the initial stage of
differentiation of ES cells via formation of EBs, and investigated
as to when neural stem cells emerged during culture. Specifically,
EBs which had undergone 4 to 8 days of culture were dissociated
into single cells, followed by culture for 7 days in a medium
designed for amplifying neural stem cells, whereby neurospheres
were formed. The neurospheres were transferred to a differentiation
medium, and allowed to differentiate. Thereafter, their
differentiation capacity was checked. Also, neurospheres were
subcultured for checking their self-renewal capacity.
FIG. 1 shows the results of selective culture of neural stem cells
(the neurosphere method), wherein 6 or 8 days after start of EB
formation through suspension culture, the formed EBs were
dissociated into single cells and subjected to the neurosphere
method. The number of the neural stem cells emerged in the EBs was
taken as that of the obtained neurospheres. Neural stem cells
(capable of forming neurospheres) which were to be identified by
the present method were virtually not detected until day 4 of
culture. On day 6 of culture, neural stem cells accounted 0.25% of
all the cells, and on day 8, neural stem cells accounted 1.1%, thus
gradual increase in cell count was acknowledged.
The neurospheres obtained from the EBs on day 6 (see FIG. 1) were
cultured for 7 days under differentiation conditions, and their
differentiation capacity was checked through immunostaining. The
results are shown in FIGS. 2 to 4. When triple immunostaining was
performed by use of .beta.-III-tubulin (a marker for neurons) and
GFAP and anti-04 antibody (markers for glial cells), virtually all
neurospheres were found to be formed only of neurons, which express
.beta.-III-tubulin, and no glial cells were detected (FIG. 2). The
neurons were found to contain at least motor neurons expressing at
least Isl-1 and ChAT (note: the motor neurons are seen in FIG. 3 as
round images and fibrous images) and GABAergic neurons expressing
GAD67 (note: the GABAergic neurons are seen in FIG. 4 as fibrous
images).
Moreover, the obtained neurospheres were subjected to subculture,
to thereby obtain secondary neurospheres. The secondary
neurospheres were cultured for 7 days under differentiation
conditions, and their differentiation capacity was checked through
immunostaining. As a result, all the neurospheres were found to
contain glia cells (FIG. 5); with 84.2% thereof containing both
neurons and glias (FIG. 6). FIG. 5 shows the results of triple
immunostaining with .beta.-III-tubulin (definite thin fibers), GFAP
(portions surrounding those of .beta.-III-tubulin), and anti-04
antibody (portions surrounding those of GFAP).
As a result, the following was confirmed: When neurospheres are
dissociated into single cells and then subcultured to thereby cause
formation of new neurospheres and differentiation, most clones
thereof contain both neurons and glias, and like the case in which
glia cells emerge in a later period in development of actual
central nervous system, neural stem cells isolated from EBs, after
undergoing subculture, also exhibit pluripotent capacity.
(2) Improvement of Efficiency in Inducing Neural Stem Cell
Differentiation by Use of Noggin Protein
In an attempt to improve efficiency in inducing neural stem cell
differentiation, during EB formation (6 days), noggin protein was
added. The noggin protein employed was in the form of solution
prepared by use of the supernatant of the culture in which
full-length cDNA of Xenopus was inserted into a pEF-BOS expression
vector and then transfected into COS7 cells for transient
expression. The control employed was a supernatant of culture of
COS7 cells to which only the expression vector had been
incorporated. As shown in FIG. 7, the number of neurospheres formed
of neural stem cells and induced to differentiate among EBs
increases with the volume of the noggin culture supernatant,
reaching a peak at 1/10 in volume.
(3) Improvement in Efficiency of Motor Neuron Differentiation by
Use of Sonic Hedgehog Protein
In an attempt to improve efficiency of motor neuron production and
differentiation from EB-derived neural stem cells, sonic hedgehog
protein was added to proliferating neural stem cells, in other
words, during formation of primary culture neurospheres derived
from EBs, and the effect of the addition was studied. After the
neurospheres were dissociated into single cells and cultured for 5
days in a differentiation medium, motor neurons were identified
through double immunostaining by use of Isl-1 and
.beta.-III-tublin, and the number thereof was quantified. As shown
in FIG. 8, production of motor neurons doubled as a result of
addition of 5 nM sonic hedgehog protein. When sonic hedgehog
protein was added to the differentiation medium in which neural
differentiation took place, no effect of addition was observed.
INDUSTRIAL APPLICABILITY
The present invention has thus found that ES cells have capability
of producing at least motor neurons and GABAergic neurons
systematically and efficiently. It also suggests that if neurons
are selectively obtained therefrom, ES cells might make it possible
to bring the potential use to transplant therapies for amyotrophic
lateral sclerosis, Huntinton's chorea, Alzheimer's disease,
etc.
* * * * *